Since I first started caring for HIV-l infected patients in 1993, I am impressed by the fact that despite the relative inefficiency of mucosal HIV-1 transmission, this mostly heterosexually transmitted global epidemic continues unabated without an effective vaccine or microbicide. One fundamental hurdle in designing prevention strategies is our lack of knowledge concerning the transmission events across the human genital mucosa. This K08 application will support my endeavor to elucidate the mechanisms of sexual HIV-l transmission while enhancing my training in molecular immunology and virology. I perceive this training as essential to accomplish my long-term research goal, which is to translate knowledge of the many facets of the human urogenital immune system and its interaction with HIV-l into strategies to interrupt HIV-l transmission. Dendritic cells (DC) and CD4+ T cells have been strongly implicated as important targets for HIV-I during sexual transmission, but direct evidence for this in the human genital mucosa is lacking. In addition, the mechanisms of viral strain selection, and particularly the preferential transmission of R5-tropic HIV-I variants, are poorly understood. We were the first to successfully isolate DC and T cells from the human genital mucosa but our studies on HIV-l coreceptor expression and in vitro infectivity did not explain viral selection during transmission. Potential pitfalls were the 36-hour long in vitroemigration procedure required for cell isolation, which may have resulted in changes of HIV-l coreceptor expression, and the inability to distinguish intraepithelial from stromal cells. To circumvent these limitations, we have established new procedures to isolate mononuclear cells from the vagina, ecto-and endocervix within three hours after surgery, and to clearly delineate cells derived from the epithelium and the underlying stroma. In Specific Aims 1 and 2 of this application, we will investigate the phenotypic and functional properties of T cells and DC infiltrating the mucosa of the human female genital tract. In Aim 1, we will compare the homing receptor profile, the state of immune differentiation and activation, and the antigen-specific repertoire of T cells among the epithelium and stroma of vagina, ecto- and endocervix, and to T cells in circulation. In Aim 2, we will define the stages of DC maturation and delineate DC lineage and differentiation pathways in the human genital mucosa. In Aim 3, we will relate the expression profile of HIV-l coreceptors on intraepithelial and stromal leukocytes isolated freshly from the genital mucosa to infectivity with R5- and X4-tropic HIV-l variants. These studies will improve our knowledge of the human genital immune system and shed light on the mechanisms of HIV-l targeting and strain selection in the female genital tract.